Figure 6. BDNF treatment of HEKTrkB cells increases GluR2-GRIP1 interaction and upregulates their total protein levels.

A) Upper panel, immunoprecipitation is done with anti-N-terminal GluR2 antibody and immunoblotting with anti-GRIP1; lower panel, immunoprecipitation is done with anti-GRIP1 and immunoblotting with anti-C-terminal GluR2/3 antibody. The results indicate increased interaction between GluR2 and GRIP1 following 1 h BDNF treatment (50 ng/mL) of HEKTrkB cells cotransfected with GluR2 and GRIP1 expression vectors. B) HEKTrkB cells are transfected with WT GluR2 and GRIP1. One day after transfection, cells are treated with BDNF or not for 1, 2, or 3 days. Protein samples are analyzed for the total protein content of GluR2 and GRIP1 as well as for phospho-GluR2 (p-GluR2-Ser880). C) Western blotting of proteins samples from cultured neurons treated with BDNF or not were run in parallel with samples extracted from HEKTrkB cells that were transfected with wild type (WT) or deletion mutant of GluR2 lacking the last 5, 10, or 21 aminoacids (Δ5, Δ10, and Δ21, respectively). The C-terminal antibody against GluR2/3 only labeled GluR2 WT. The N-terminal anti-GluR2 labeled all constructs. D) HEKTrkB cells were transfected with GluR2Δ5 together with GRIP1. The results indicate gradual accumulation of WT GluR2 but not GluR2Δ5 in samples treated with BDNF.